(a) Field of the Invention
This invention relates generally to a novel method for quantifying the number of occurrences of a specific nucleic acid sequence within a nucleic acid sample in order to circumvent the shortcomings of the methods currently available and to provide reliable quantification of a specific nucleic acid sequence within a nucleic acid sample.
(b) Description of Prior Art
DNA Quantification
Biological processes are governed, in part, by the presence of specific DNA sequences present in the genome of living organisms. Their relative quantities or respective expression levels will contribute to define cellular functions and to determine the phenotypical traits of the organism.
Considerable effort has been devoted to the development of techniques for quantifying the number of times a particular sequence occurs in a genome. One such technique is Southern hybridization (Southern, E. M., 1975, J. Mol. Biol., 98: 503-517). Briefly, DNA extracts are enzymatically digested, resolved by gel electrophoresis, transferred onto a solid support (nitrocellulose or nylon membranes) and probed with labeled poly-nucleic acids. The results show a number of bands representing restriction fragments onto which the labeled probe hybridized. The number and/or intensity of bands can be used to determine the number of copies of the targeted DNA fragment per haploid genome. This technique has the disadvantage of being time consuming and poorly adaptable to a high-throughput production environment. Also, if the labeled probe hybridizes to two restriction fragment of roughly the same size, the resulting band will have a higher intensity the other bands, if present. Therefore, in such cases, the intensities of the bands will have to be determined by other means in order to get good estimates of the number of copies of the target sequence.
Copy number determination can also be achieved by the polymerase chain reaction technique (Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G. & Erlich, H., 1986, Cold Spring Harbor Symp. Quant. Biol., 51: 263-273). After amplification, the PCR products are resolved onto agarose gels and visualized using DNA-specific fluorescent dyes such as ethidium bromide. The DNA-dye complex fluorescent intensity of the target DNA sequence is then compared on the agarose gel to the intensity obtained with control DNA of known concentrations. Also, the PCR amplification can be designed in such a way that two PCR products are amplified in the same reaction tube (duplex PCR). One PCR product will be specific to the target sequence and the other one will serve as a control and is constant between the two samples. The DNA-dye complex fluorescent intensity for the target sequence is then compared to the intensity obtained with the control PCR product. In both cases, this technique will produce more qualitative than quantitative estimates of the actual initial number of copy of the target sequence.
Variants of the polymerase chain reaction can be used to determine the initial number of copies of a specific target sequence. Competitive PCR is based on the principle that if two DNA fragments sharing oligonucleotide binding sites are amplified in a duplex PCR reaction, they will compete for the availability of primers. After amplification, the relative quantity of the two amplicons will be used to determine the starting number of copies for each amplicon. This technique requires that a competitor DNA be added to the genomic (sample) DNA extracts in different concentrations, and therefore has no endogenous control. It also requires that the competitor DNA sequence is different from the target DNA sequence, in most cases in length, to make it possible to distinguish it from the DNA fragment under investigation. It is then possible to determine, by gel electrophoresis for example, with which concentration of competitor DNA the amplification products were amplified in comparable amounts. This technique also requires that the concentrations of the DNA extracts be standardized prior to these quantifications in order to make comparisons between samples possible.
Real-time PCR is a method that measures amplicon quantities during the PCR amplification. This is generally achieved by adding one or two labeled oligonucleotides bearing a fluorochrome and a quencher to the PCR cocktail, (example: TaqMan assay). Fluorescence is monitored throughout the amplification and is emitted only if the two labels are not in close vicinity to each other when hybridized to one strand of the amplicon. By using fluorochromes emitting at different wavelengths linked to oligonucleotides specific to more than one amplicon, it is possible to monitor different amplicon quantities in a single tube. This allows for the possibility of amplifying an endogenous control and achieving reliable quantification of the target sequence. Real-Time PCR is particularly useful when a wide range of resolution is needed (e.g. 10 vs. 10,000 copies), but is limited in resolution when differentiating discrete variations, e.g. two, three, four or five fold more copies in one sample as compared to another one.
Potential Applications of DNA Quantification
In the process of creating genetically modified organisms (GMO), the gene conferring the novel trait is transferred into the genome of the target cells. These transformed organisms have to go through a registration process before they can be released for commercial use. For this purpose, it is necessary to select the transformants in which a single integration event took place. If the GMOs were created in order to elucidate the function of a gene or its phenotypical effects, results drawn will be more conclusive if the transgene is present only once in the genome of the transformed organism and expressed in a similar fashion as it is in its endogenous location in the source organism.
Some genes have redundant functions, meaning that two or more genes, located in separate locations in the genome, might have identical functions. The number of such paralogous genes could be determined with techniques described herein. In addition, these techniques could be used to determine if the copy number of one or more of the cell's chromosomes, or parts of chromosomes, is abnormal. For example, when cells are grown in culture, chromosomal aberrations, such as the duplication or loss of chromosomes or parts of chromosomes can occur. Such abnormalities can also occur after treatment of with mutagens.
Certain diseases are associated with abnormal ploidy. Ploidy refers to the number of copies of each individual chromosome in a cell; for most non-reproductive cells in animals and plants, this number is two (the diploid chromosome number). Under certain conditions, in some types of human tumor cells for example, this number may be greater than two for some or all chromosomes, suggesting that genetic mechanisms that maintain chromosome copy number have been disrupted. Such disruptions can be seen in prostate, ovarian and breast cancer. Studies have shown that patients with diploid cancers (having normal DNA content in cancer cells) have longer cancer-free intervals and survival than those with non-diploid cancers. Diploid tumors are also more responsive to hormonal therapy.
Ploidy counts can sometimes be used to identify specific tissues, for example, in plants. The endosperm of seeds is triploid and other tissues are diploid. Anther culture is a method for producing haploid plants that can be used for the development of double haploid populations. There have been reports of ploidy variations in anther culture of barley (Sunderland, N. (1980) In: The Plant Genome, Proc. 2nd Haploid Symp. John Innes Inst. Norwich, pp 171-183) and unexpected ploidy variation among progeny plants in wheat, due to gametoclonal and somoclonal variations (Baenziger et al. (1989) Plant Breeding 103:53-56).
RNA Quantification
Considerable efforts have also been made to develop techniques to quantify relative amounts of RNA in extracts in order to estimate the level at which a gene is transcribed. One method for detecting the expression of a gene is Northern blotting. The principle of this technique is basically the same as for Southern blotting; purified RNA is resolved on an agarose gel in denaturing conditions, transferred onto a solid support and probed with a labeled nucleic acid sequence. The intensity of the signal corresponding to the probe hybridized to the target sequence will reflect expression level. A relative estimation of the level of expression can be obtained by comparing the intensity of the band between two RNA extracts that have been normalized by a second probe that hybridizes to an RNA that is expressed at the same level in the two extracts. When a small part of the RNA is degraded, the bands produced by the labeled probes are of unequal sizes and smeared, depending on the level of degradation. Calculating the intensity of the probe's signal on bands of different sizes will produce only a qualitative estimate, not reliable quantitative expression level data. Also, being based upon hybridization, this technique does not have a high enough resolution to discriminate the expression levels of highly identical genes. Finally, the technique is lengthy and labor intensive.
The newly developed micro-array or DNA-chip technology can also be used to monitor expression levels of genes. Polynucleotide molecules (oligonucleotides, cDNA or genomic sequences) are deposited on a solid support (glass slide, nylon or nitrocellulose paper) as homogenous dots (one dot contains polynucleotides that all share the same sequence). They are then probed with complex mixtures of labeled cDNAs or RNA. The probes and their complementary sequences deposited on the solid support hybridize and the relative quantification of expression levels of the genes can be determined by measuring the quantity of labeled probes specific to a homogenous spot. As it is the case for Northern hybridization, this technique does not have a high enough resolution to discriminate the expression levels of highly identical genes.
RNA extracts can also be treated with an enzyme called reverse transcriptase to generate corresponding DNA sequences. After such treatment, copy number or expression level analyses will be performed as it is done with DNA extracts.
It would be highly desirable to be provided with a novel method for quantifying the number of occurrences of a specific nucleic acid sequence within a nucleic acid sample in order to circumvent the shortcomings of the methods currently available and to provide reliable quantification of a specific nucleic acid sequence within a nucleic acid sample.